Human embryonic stem cells (hESC) are isolated from the inner cell mass of an early stage human embryo1-4. They are distinguished by their ability to replicate indefinitely (self-renewal)5-8 and under the appropriate conditions differentiate into cells of all three germ layers (ectoderm, endoderm and mesoderm)4,9,10. Mounting studies have reported the successful differentiation of hESC into embryoid bodies (EBs)11 and several lineage-specific cell types, such as cardiomyocytes12-16, hepatocytes17, neurons18-22, endothelial cells 23-28, osteoblasts29,30, keratinocytes31 and retinal pigment epithelium (RPE)32,33. Their successes demonstrate the great potential of hESC in tissue engineering and regenerative medicine to treat various diseases such as diabetes, heart failures, Parkinson's disease, degenerative eye diseases, and skeletal tissue injuries. However, tumorigenicity, as a major safety concern, is still impeding the progress of hESC-based therapies34-36. Briefly, tumorigenicity of hESC refers to the formation of teratomas (benign tumors) or even teratocarcinomas (malignant tumors) in the differentiated cell products due to the presence of residual undifferentiated hESC.
Previously, a panel of monoclonal antibodies (mAbs) has been generated against hESC to characterize hESC populations and discover novel hESC surface markers37. See WO2007/102787 and WO2010/033084. Among these antibodies, mAb84 was firstly found to be able to kill undifferentiated hESC. However, since mAb84 is an IgM pentamer, its relatively large molecule size and tendency to form aggregates might impede its penetration efficiency into tumor mass. Subsequently, a smaller antibody fragment format of mAb84, scFv84-HTH, was engineered and demonstrated to have improved penetration by Lim et al.38. However, to achieve the same level of cytotoxicity on hESC, the amount of scFv84-HTH required is 20 times more than mAb84. Moreover, since mAb84 and scFv84-HTH are either multivalent or bivalent, the author speculated that antibody cytotoxicity is associated with its valency.
There are several strategies36,37 to prevent tumorigenicity of hESC, which can be categorized into 3 stages. First, in the pre-transplantation stage, hESC can either be terminally differentiated into the desired cell types or undifferentiated cells can be removed from differentiated cells products by various sorting techniques. Second, in the early post-transplantation stage, tumor progression can be interrupted with methods such as genetic manipulation or cytotoxic drugs. Third, in the late post-transplantation stage, detected tumors carrying an engineered “suicide gene” can be eliminated by drugs. Various techniques to eliminate undifferentiated hESC or tumor formation at different stages, as well as their advantages and disadvantages are summarized in FIG. 1. However, none of these methods is capable of completely precluding teratomas or teratocarcinomas formation in vivo.
Applications of cytotoxic antibodies are diverse. Firstly, cytotoxic antibody-induced cell death can be used as a cell model to study molecule and cellular functions. In 1995, Bazil et al. generated a monoclonal antibody, MEM-59, which recognizes the surface adhesion molecule CD43 of human hematopoietic progenitor cells (HPCs) and directly kill HPCs via cross-linking of CD4361. With MEM-59 induced-HPCs death, CD43 was identified as a negative regulator of early hematopoietic events. Secondly, cytotoxic antibodies can be used to identify novel pathways of cell death and to characterize cell death. Matsuoka et al. has generated a monoclonal antibody, RE2, which could induce a novel type of cell death of activated interleukin 2-dependent T cells62. Zhang et al. also found a cytotoxic antibody, anti-Porimin, towards Jurkat cells63. In this study, they introduced the very first cell surface receptor-mediated pathway of cell death as well as some unique features of cellular response upon cell death, such as cell aggregation, plasma membrane permeabilization and membrane blebs. Thirdly, as mentioned in the previous section, cytotoxic antibodies have been used intensively to eliminate undesired cells, such as the treatment of cancers. Rituximab, a monoclonal antibody targeting the CD20 antigen on B-lymphocytes, was the first cytotoxic antibody approved to treat B-cell malignancies, such as non-Hodgkin's lymphoma, in combination with chemotherapy64. Rituximab induces cell death upon hyper-cross linking with goat anti-human secondary antibody after 20 h of incubation. To overcome the rituximab-resistance in some patients, many next-generation anti-CD20 cytotoxic antibodies have been generated65,66. GA101 is a novel anti-CD20 cytotoxic antibody, which induces B-lymphocyte death by dispersing lysosomes contents into the cytoplasm and surrounding environment67. There are also other cytotoxic antibodies such as RAV12 that induces recurrent adenocarcinoma cell death68 and anti-NeuGcGM3 antibodies that directly kill lung cancer cells69,70. Although all these cytotoxic antibodies can induce cell death, the modes of cell death are varied (FIG. 2).
For a long-time, programmed cell death has been used synonymously with apoptosis and oncosis has been considered as accidental cell death. However, this concept was proved to be not always true71, where oncosis can also be a pattern of programmed cell death. In fact, every cell may experience “programmed” cell death upon an appropriate stimulus, whereas the pattern (apoptosis or oncosis) differs among cell types and the stimulus72,73. In general, cell undergoes three phases upon lethal injuries72:                a) Reversible “pre-mortal phase”;        b) Irreversible cell death, “point-of-no-return”;        c) Post-mortal autolytic and degradative changes.        
One way to distinguish different types of cell death and its terminology is to define them by cellular response in each phase of cell death. In the pre-mortal phase, there are two major modes of cell death: apoptosis and oncosis. They are mainly distinguished by cell volume alteration and cellular morphological changes. The hallmarks of apoptosis and oncosis will be discussed in detail later. On the contrary, most of post-mortal cellular changes were termed “necrosis”72, which is an ancient word describing cellular changes after cell death.
Apoptosis was first proposed by Kerr et al. in 1972 to describe a pattern of controlled cell deletion. In the regulation of normal development and cell population, apoptosis was thought to play a complementary but opposite role to mitosis74. Dysregulation of apoptosis would result in many diseases such as cancer, Alzheimer's and autoimmune diseases75,76.
The process of apoptosis is tightly controlled and organized. It is characterized by a set of morphological changes such as cellular shrinkage, nuclear chromatin condensation and budding of plasma membrane, and biochemical changes such as protein cleavage, cross-linking, patterned DNA fragmentation and phagocytic recognition77. Apoptosis usually begins with caspases activation 12 to 24 hours after a trigger event. Changes in plasma membrane protein and cytoskeleton would result in the formation of apoptotic bodies, which enclose fragments of nucleus and cell organelles by intact plasma membrane74,78. Subsequently, apoptotic bodies with phosphatidylserine expressed on the outer membrane are recognized and engulfed by the neighboring cells, in particular macrophages and endothelial cells. Eventually, cell debris is cleared out from the tissue to avoid inflammatory response78. Generally, there are three major pathways to caspase activation: extrinsic caspase-8 activation via receptor-ligand binding, intrinsic caspase-9 activation via mitochondria and caspase-12 activation via endoplasmic reticulum79. To distinguish apoptosis from other modes of cell death, the hallmarks of apoptosis and respective assays are summarized in FIG. 3.
Oncosis was first proposed by Von Rechkling-hausen in 1910 to describe cell death with swelling and later used to describe ischemic cell death distinct from apoptosis80. After injury, oncosis can be triggered within seconds to minutes followed by marked cell shape and volume alteration in early stage72. It was characterized by several morphological and biochemical changes such as apparent swelling of cell and organelles, gross vacuolization, membrane permeabilization and cytoskeleton proteins degradation72,80. Comparing to the understanding of apoptosis, the mechanism of oncosis is still under investigation. Some studies have shown that failure in the ionic pumps of the plasma membrane and decreased levels of cellular ATP might be the cause of oncosis73,80. With current understanding, oncosis can be detected by identifying their hallmarks with respective assay, as summarized in FIG. 4A.
In general, apoptosis and oncosis are pre-mortal process, which can lead to post-mortal necrosis. After the phase of apoptosis and oncosis, changes are similar in the phase of necrosis, termed apoptotic necrosis or oncotic necrosis.
As described above, cytotoxic antibodies can induce cell death. Studies on some of these cytotoxic antibodies have shown that cells undergo different modes of cell death (apoptosis or oncosis) upon incubation with cytotoxic antibodies (FIG. 2).
MEM-59 was shown to induce apoptosis in hematopoietic progenitor cells as cell shrinkage and DNA fragmentation were detected61. Another antibody, Rituximab, also induces non-Hodgkin's lymphomas apoptosis after 18 to 20 hours incubation. Other detected apoptosis hallmarks including DNA fragmentation, phosphatidylserine exposure detected by Annexin V, and increase in caspase-3 activity64.
There are also antibodies that induce oncosis in cells. Rapid swelling of RAV12-treated adenocarcinoma cells was observed within 1 hour81. Under time-lapse microscopy, membrane damage was detected followed by cell swelling. In addition, disruption of actin cytoskeleton and elevated LDH were also observed upon RAV12 treatment. Another example of antibody-induced oncosis is anti-Porimin63. Increase in membrane permeability of anti-Porimin treated Jurkat cells was detected by PI uptake. Moreover, formation of pores on the cells membrane was also visualized under Scanning Electron Microscope (SEM). Another hallmark of anti-Porimin induced oncosis is the re-arrangement of cytoskeletal proteins. A more relevant example is mAb84-induced hESC death via oncosis. Studies have also shown some oncotic features of mAb84-induced hESC death, such as rapid cell death, formation of cell aggregation, loss of membrane integrity and degradation of actin cytoskeleton-associated proteins82. However, for all mentioned studies, though the mode of cell death was identified, the detailed mechanism of antibody-induced cell death is still unknown.
WO2012/011876 describes a method of selecting an antibody as a candidate for having cytotoxic activity against a cell which expresses podocalyxin-like protein (PODXL) where the antibody binds PODXL, the method comprising a step of comparing the binding of a PODXL-binding antibody molecule to a glycan comprising Fucα1-2Galβ1-3GlcNAc with the binding of a non-cytotoxic PODXL-binding antibody molecule to a glycan comprising Fucα1-2Galβ1-3GlcNAc.